Whole Genome Amplification (WGA) of genomic DNA has been previously described. The methods have generally been based on Degenerate Oligonucleotide Primed PCR (1992 Telenius, H, et al, Genomics, 13, 718-725). Although these techniques have been shown to produce some degree of random genomic amplification, the methods have been limited in the size of the DNA fragments amplified. Using Taq polymerase (Taq) amplified representations have generally been limited to fragments varying in size from 100-2000 bp thus limiting the usefulness of these methods. By using cocktails that contain a polymerase and an additional proof reading enzyme, larger fragments up to 10 kb in size have been reported. The use of proof reading polymerase enzymes on bisulphite-treated DNA, however, generally yield no amplification due to the inherent proof reading capabilities of these polymerases which do not recognize the uracil bases in bisulphite-modified DNA (Sakaguchi, A. Y., et al Biotechniques. 1996 September; 21 (3):368-70.).
More recently, another method for WGA has been described. This method, termed Rolling Circle Amplification (RCA), is an isothermal amplification strategy employing the enzyme phi29 (2002 Dean et al, PNAS, 99(8), 5261-5266). This method does not depend on denaturation of template molecules at every cycle of the reaction unlike conventional amplification technologies. The enzyme phi29 will continually produce a DNA amplification product at 30° C. via a rolling circle method of amplification (2002 Dean et al, PNAS, 99(8), 5261-5266). In addition, using this method on normal DNA, primers as short as 6 bp have been found to be adequate for WGA. However, the phi29 enzyme has inherent exonuclease activity, thus enzyme resistant primers must be used. Using this strategy, amplified products in excess of 30 kb in size have been achieved from normal DNA (2002 Dean et al, PNAS, 99(8), 5261-5266).
The unique nature of bisulphite-modified DNA, (in which the complementary strands become different in sequence after bisulphite treatment), means that traditional methods for WGA cannot be applied. This is due to the fact that after bisulphite treatment the two DNA strands are no longer complementary. Thus to perform WGA on bisulphite-treated DNA, at least 2 primers of different sequence have to be added to an amplification reaction to target each strand of the DNA, rather than the one that would be used for amplification of normal DNA.
In addition conventional bisulphite treatment of DNA has been shown to result in the loss of up to 96% of the starting DNA (2001, Grunau C et al, Nucleic Acids Research, 1:29(13):E65-5). The use of such DNA would not be suitable for the use in WGA methods as most of the DNA has been lost therefore any representation obtained would not be a true reflection of the original starting material.
The present inventor has now developed methods for WGA that can be used to amplify genomic DNA that has undergone bisulphite treatment.